Arch. Pharm. Res. DOI 10.1007/s12272-015-0592-9
RESEARCH ARTICLE
Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats Tae-Heon Kim1 • Jong-Woo Jeong1 • Ji-Hye Song1 • Kyeong-Ryoon Lee2 Sunjoo Ahn3,4 • Sung-Hoon Ahn3,5 • Sungsub Kim1 • Tae-Sung Koo1
•
Received: 1 September 2014 / Accepted: 30 March 2015 Ó The Pharmaceutical Society of Korea 2015
Abstract We characterized the pharmacokinetics of enzalutamide, a novel anti-prostate cancer drug, in rats after intravenous and oral administration in the dose range 0.5–5 mg/kg. Tissue distribution, liver microsomal stability, and plasma protein binding were also examined. After intravenous injection, systemic clearance, volumes of distribution at steady state (Vss), and half-life (T) remained unaltered as a function of dose, with values in the ranges of 80.4–86.3 mL/h/kg, 1020–1250 mL/kg, and 9.13–10.6 h, respectively. Following oral administration, absolute oral bioavailability was 89.7 % and not dose-dependent. The recoveries of enzalutamide in urine and feces were 0.0620 and 2.04 %, respectively. Enzalutamide was distributed primarily in 10 tissues (brain, liver, kidneys, testis, heart, spleen, lungs, gut, muscle, and adipose) and tissue-to-plasma ratios of enzalutamide ranged from 0.406 (brain) to 10.2 (adipose tissue). Further, enzalutamide was Tae-Heon Kim and Jong-Woo Jeong have been contributed equally to this work. & Sungsub Kim [email protected] & Tae-Sung Koo [email protected] 1
Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon, Korea
2
Life Science Research Institute, Daewoong Pharmaceutical Corporation, Yongin, Korea
3
Center for Drug Discovery Technology, Korea Research Institute of Chemical Technology, Daejeon, Korea
4
Department of Medicinal Chemistry & Pharmacology, Korea University of Science and Technology, Daejeon, Korea
5
College of Pharmacy, Kangwon National University, Chuncheon, Korea
stable in rat liver microsomes, and its plasma protein binding was 94.7 %. In conclusion, enzalutamide showed dose-independent pharmacokinetics at intravenous and oral doses of 0.5–5 mg/kg. Enzalutamide distributed primarily to 10 tissues and appeared to be eliminated primarily by metabolism. Keywords Enzalutamide Pharmacokinetics Tissue distribution Metabolsim Excretion
Introduction Prostate cancer is the most common malignancy and the second leading cause of cancer-related death in men worldwide. The World Health Organization (WHO) has reported the worldwide prostate cancer prevalence as *900,000 diagnosed cases per year, and 239,000 new cases, in the United States in 2013, with a projected death total of 29,700 (Parkin et al. 2005). Prostate cancer develops in the prostate gland of the male reproductive system. The growth of prostate cancer has been demonstrated to be involve androgens and androgen signaling pathways (Huggins and Hodges 2002). Generally, surgical and chemical castration have been used to treat prostate cancer. While surgical castration includes radical orchiectomy (i.e., the removal of the testicles, the source of 95 % of the body’s testosterone), chemical castration may involve administration of various pharmaceuticals such as luteinizing hormone-releasing hormone (LH-RH) agonists, anti-androgens, or other testosterone biosynthesis-inhibiting drugs. Application of androgen deprivation therapy (ADT) may lead to decreased testosterone biosynthesis and result in a reduction in prostate-specific antigen (PSA), tumor degeneration,
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remedy of symptoms, and increased survival of patients (Crawford et al. 1989). Although considerable progress has been made in improving the therapy of prostate cancer, one-third of treated prostate cancer patients will experience disease recurrence and will progress into castration-resistant prostate cancer (CRPC). For these reasons, there is a continuing need for novel drugs that may selectively target the major signaling pathways that contribute to CRPC. Because androgen receptor (AR)-associated pathways are responsible for cancer cell growth and survival, and even enable the cancer cells to grow in the presence of minimal androgen levels (Kahl et al. 2006; Metzger et al. 2005), AR signaling is a useful target for designing and developing such drugs. However, many first-generation antiandrogens, such as flutamide, nilutamide, cyproterone acetate, and bicalutamide, have failed to demonstrate substantial efficacy in CRPC patients. Moreover, these drugs may have AR-agonizing effects (Nguyen et al. 2007). Enzalutamide, previously known as MDV3100 and now available commercially as XtandiÒ, is an anti-prostate cancer drug that is a second-generation AR signaling inhibitor. Enzalutamide blocks the growth of CRPC in cellular model systems and has been shown to increase survival in patients with metastatic CRPC (mCRPC) in a clinical trial (Semenas et al. 2013). Enzalutamide was recently approved (2012) for the treatment of mCRPC postdocetaxel by the US Food and Drug Administration (Mullard 2013; Semenas et al. 2013). Unlike antiandrogens (e.g., bicalutamide), enzalutamide does not have agonist effects in AR signaling but competitively inhibits androgen binding to the AR. Moreover, it inhibits the nuclear translocation of androgens and interactions with DNA. As a result, it induces apoptosis and decreases tumor volume and the proliferation of cancer cells (Scher et al. 2012; Tran et al. 2009). Some clinical pharmacokinetic studies on enzalutamide have been reported (Astellas 2012; El-Amm et al. 2013; Scher et al. 2010), although there limited information describing the pharmacokinetics of enzalutamide in animal models (Astellas 2014; Song et al. 2014). The pharmacokinetics in experimental animals, especially rats, is generally useful as reference data for drug discovery and basic data for analyzing efficacy and toxicity. Also, the evaluation of pharmacokinetics is meaningful for understanding the specific properties (e.g., absorption, distribution, metabolism, and elimination) of drugs in the body. Thus, in the present study, we characterized the pharmacokinetics of enzalutamide after intravenous and oral administration, and evaluated its distribution into major tissues, including the brain, heart, liver, kidney, and testis, in rats.
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Materials and methods Chemicals Enzalutamide was obtained from the Korea Research Institute of Chemical Technology, and bicalutamide, an internal standard, was purchased from Sigma Aldrich. Ammonium acetate salt and other reagents were also from Sigma Aldrich. Acetonitrile and methanol were HPLCgrade and from J.T. Baker. Rat plasma was anticoagulated with sodium heparin, and came from our laboratory. Animals Sprague Dawley (SD) rats (males, 7 weeks old, 204–230 g) were purchased from Orient Bio Inc., (Seongnam, Korea). All animals were kept in an animal room with controlled illumination (12/12 h light/dark cycle), temperature (20–25 °C), and humidity (over 40 %). The animals were fasted for 15 h before dose administration. Animal experiments Intravenous and oral administration of enzalutamide in rats Enzalutamide was dissolved in vehicle (10 % DMSO, 45 % polyethylene glycol 400, and 45 % saline). As a single dose, the administration routes were an intravenous bolus via the tail vein (n = 3) and an oral gavage dose (n = 4). Dosing volume was 1 mL per kg (body weight) and the dosing range was 0.5, 2, and 5 mg/kg. The use of 2 mg/kg was reported in a previous study (Song et al. 2014). A blood sample (200 lL) was collected from the jugular vein using a heparinized syringe to ensure anticoagulation at 0.08 (intravenous only), 0.33, 1, 3, 6, 10, 24, 48, and 72 h after dosing. During blood sampling, rats were placed in a restrainer (Nagai-CFS-1S, NMS, Tokyo, Japan) (Koo et al. 2011). To prepare plasma samples, all blood samples were centrifuged at 13,5009g for 5 min. The samples were stored at -20 °C until analyzed. Determination of urinary and fecal excretion Male SD rats (n = 3) were administered enzalutamide through the tail vein (intravenous) and by oral gavage at 1 mg/kg and were kept in metabolic cages after dosing. Urine and feces samples were collected over the following time intervals after dosing: 0–2, 2–4, 4–6, 6–10, 10–24, 24–48, and 48–72 h. The metabolic cages were rinsed with distilled water, and residues were added to the urine samples at 72 h. To extract the enzalutamide present in the
Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats
feces, samples were shaken vigorously for 12 h with 50 % methanol. The amount of drug excreted in urine or feces (Ae) and the fractions of dose excreted unchanged in urine or feces (fe) were calculated as: Ae ¼Cobs; urine or feces Vurine or feces fe ¼Ae =Dose The urinary and fecal clearances of enzalutamide were calculated as: CLurine or feces ¼ Ae; urine or feces =AUC Estimation of tissue-to-plasma concentration ratio Male SD rats were administered enzalutamide as an intravenous bolus (n = 3, at each time point); dosing conditions were 1 mg/kg (in 10 % DMSO, 45 % polyethylene glycol 400, and 45 % saline) and 1 mL/kg. Animals were sacrificed at 0.08, 0.33, 1, 3, 6, 10, 24, 48, and 72 h after dosing. As biological materials, blood and 10 major tissues (brain, testis, gut, liver, kidney, spleen, lung, adipose, muscle, and heart) were collected. Each tissue was homogenized with the same weight of water (50:50, w/w) using a homogenizer for 5 min at 30 Hz (FB50, Fischer Scientific, USA). The plasma samples and tissue homogenates were stored at -20 °C until analyzed.
In vitro experiments Estimation of hepatic intrinsic clearance of enzalutamide in rat liver microsomes Rat liver microsomes (BD Biosciences, San Jose, CA) were used to estimate the intrinsic clearance of enzalutamide. A typical reaction mixture (500 lL) consisted of rat liver microsomal protein (final concentration = 0.5 mg protein/ mL incubation mixture) and an NADPH regenerating system (final concentrations: 1.3 mM NADP?, 3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, and 3.3 mM magnesium chloride) in 100 mM potassium phosphate buffer (pH 7.4). The mixture was pre-incubated in a water bath at 37 °C for 5 min and an aliquot of enzalutamide solution added to a final concentration of 2 lM. Aliquots (50 lL) of the mixture were sampled at 0, 5, 15, and 30 min after initiation of the reaction. Immediately after collection, a stop solution (100 lL ice-chilled methanol) was added to the sample to terminate the reaction. After vigorous vortexing, and centrifuging at 10,0009g for 5 min, an aliquot (50 lL) of the supernatant was assayed. The concentration of enzalutamide remaining in the sample was plotted against the
reaction time to determine the metabolic rate constant of the reaction. To estimate the in vitro intrinsic clearance in rat livers, that clearance was calculated using the metabolic stability data from rat liver microsomes. The slope (ke) of the temporal profile for the percentage of enzalutamide remaining in a microsomal incubation were obtained by linear regression after logarithmic transformation. Assuming that the substrate concentration used in the experiments (2 lM) was below the KM for the metabolic reaction, the in vitro intrinsic clearance (CLint, in vitro) was calculated as: CLint; in vitro ¼ Aprotein ke =Cprotein where Aprotein is the total amount of liver microsomal protein, and Cprotein the microsomal protein concentration (mg/mL). In this calculation, an Aprotein of 1790 mg protein per kg was used for the rat (Houston and Carlile 1997). Estimation of the fraction of enzalutamide bound to plasma protein We conducted a protein-binding study to determine the fraction of unbound enzalutamide in rat plasma. Binding of test material was assessed via equilibrium dialysis using REDÒ devices (Thermo, Rockford, IL, USA). All assessments were made in triplicate. After 200 lL samples of plasma containing 2 lg/mL enzalutamide were placed into a sample chamber, 350 lL of phosphate buffer (pH 7.4) was added to the buffer chamber. The device containing the samples was incubated at 37 °C for 4 h in a shaking water bath. After incubation, enzalutamide in was assayed in plasma and buffer. Analytical procedure for the determination of enzaluta mide To evaluate enzalutamide levels in the biological samples, a specific high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) (FDA 2001; Song et al. 2014) protocol was used. To induce the precipitation of endogenous proteins, an aliquot (50 lL) of internal standard (IS) solution (bicalutamide 1 lg/mL in acetonitrile) and acetonitrile (200 lL) was added to the biological materials (50 lL; plasma, urine, feces or tissue homogenate). The mixture was mixed vigorously for 10 min, followed by centrifugation (13,5009g, 15 min). An aliquot (5 lL) of the supernatant was injected directly into the HPLC/MS/MS system. The HPLC/MS/MS system consisted of an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) and an API 4000 tandem quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City,
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CA, USA). Compounds were separated on an Agilent Eclipse Plus C18 column (3.5 lm, 2.1 9 50 mm; Agilent Technologies) with an isocratic mobile phase comprising 10 mM ammonium acetate in 40:60 (v/v) water–methanol at a flow rate of 0.3 mL/min. The column and autosampler tray were held at 25 and 4 °C, respectively. The eluent was introduced directly into the tandem quadrupole mass spectrometer through a turbo ion spray source with the following settings: curtain gas, 20 psi; nebulizer gas, 40 psi; turbo gas, 40 psi; ion spray voltage, 5500 V; temperature, 500 °C; declustering potentials, 96, 91 and 101 V; entrance potential, 10 V; collision energies, 47 and 51 V; collision cell exit potentials, 14 and 16 V for EZT and IS, respectively. The analytical run time was 5.0 min. Multiple reaction monitoring (MRM) mode was used for quantification at m/z 465 ? 209 for EZT and m/z 431 ? 217 for the IS. Data were analyzed using Analyst software (version 1.4.1; Applied Biosystems/AB Sciex). The quantification limits for enzalutamide in rat plasma, urine, feces and tissue homogenate were 1, 1, 3 and 3 ng/ mL, respectively. The coefficients of variation of the assay (within and between-day) were generally low (below 10.6 %).
(ANOVA), with Tukey’s test. For the determination of statistically significant correlations, tests of zero correlation were used. In both analyses, p values \0.05 were considered to indicate statistical significance (Fig. 1).
Results Figure 2 describes the mean plasma concentration–time profiles of enzalutamide after intravenous bolus injection of doses in the range 0.5–5 mg/kg in rats (n = 3). The calculated pharmacokinetic parameters are shown in Table 1. The AUC0–? values were 5.85 ± 0.67, 24.2 ± 6.2, and 63.7 ± 12.8 lg h/mL for the 0.5, 2, and 5 mg/kg doses, respectively. The AUC increased linearly as the dose increased. The MRTs were 14.2, 12.0, and 14.7 h for the 0.5, 2, and 5 mg/kg doses, respectively. The CLs were 86.3 ± 10.7, 86.2 ± 19.7, and 80.4 ± 14.5 mL/h/kg for doses of 0.5, 2, and 5 mg/kg, respectively. Vss values were 1250 ± 420, 1020 ± 110, and 1140 ± 70 mL/kg for doses of 0.5, 2, and F F3C
Pharmacokinetic analyses
NH
S
NC
N
N
O Fig. 1 Chemical structure of enzalutamide
Plasma concentration of enzalutamide (µg/mL)
The areas under the plasma concentration–time curve (AUC) and the first moment curve (AUMC) were calculated using the linear trapezoidal method, extrapolated to time = infinity. The terminal half-life (T) was calculated as 0.693/k, where k is the slope of the log-linear portion of the concentration–time profile. The systemic clearance (CL), mean residence time (MRT), and the volume of the distribution at steady state (Vss) were calculated as dose/ AUC, AUMC/AUC, and MRTCL, respectively. The extent of absolute oral bioavailability (F) was estimated by dividing the AUC after oral administration by the AUC after intravenous administration of the respective dose. The peak concentration (Cmax) and the time to reach Cmax (Tmax) were read directly from individual plasma concentration–time profiles. The tissue-to-plasma partition coefficient (Kp) for enzalutamide was calculated by dividing the mean AUCtissue by the mean AUCplasma after administration. To obtain the pharmacokinetic parameters above, all plasma and tissue concentration–time profiles were analyzed using a non-compartmental method with nonlinear least squares regression using the WinNonlin software (ver. 4.1; Pharsight, St. Louis, MO).
O
10 5 mg/kg Intravenous dose 2 mg/kg Intravenous dose 0.5 mg/kg Intravenous dose 1
0.1
0.01
0.001 0
8
16
24
32
40
48
56
64
72
Time (h)
Statistical analyses All data are presented as means ± SDs. Statistical analyses were performed using one-way analysis of variance
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Fig. 2 Plasma concentration–time profiles for enzalutamide in rats after intravenous bolus injection. Each data point represents the concentration of enzalutamide after the intravenous injection of doses ranging from 0.5 to 5 mg/kg
Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats Table 1 Pharmacokinetic parameters of enzalutamide after intravenous injection
Table 2 Pharmacokinetic parameters of enzalutamide after oral administration
Parameter
Parameter
Dose (mg/kg) 0.5
2
a
5
Dose (mg/kg) 0.5
2a
5
AUC (h lg/mL)
5.85 ± 0.67
24.2 ± 6.20
63.7 ± 12.8
AUC (h lg/mL)
5.04 ± 0.56
17.3 ± 1.0
51.8 ± 5.3
T (h)
10.6 ± 2.10
9.13 ± 0.56
10.2 ± 2.2
T (h)
10.4 ± 1.5
8.40 ± 2.46
8.96 ± 1.42
CL (mL/h/kg) MRT (h)
86.3 ± 10.7 14.2 ± 2.9
86.2 ± 19.7 12.0 ± 1.7
80.4 ± 14.5 14.7 ± 3.9
Tmax (h) Cmax (lg/mL)
2.33 ± 3.13 0.28 ± 0.04
4.00 ± 2.00 0.99 ± 0.20
6.25 ± 4.27 2.62 ± 0.76
Vss (mL/kg)
1250 ± 420
1020 ± 110
1140 ± 70
F (%)
86.2
71.8
Data are presented as means ± SDs (n = 3)
Data are presented as means ± SDs (n = 4)
a
a
Data from Song et al. (2014)
Plasma concentration of enzalutamide (µg/mL)
5 mg/kg, respectively, and T values were 10.6 ± 2.1, 9.13 ± 0.56, and 10.2 ± 2.2 h for doses of 0.5, 2, and 5 mg/kg, respectively. There were no significant differences in CL or Vss among the doses (one-way ANOVA), indicating that the elimination and distribution of enzalutamide were linear processes. The plasma concentration–time profiles after oral administration from 0.5 to 5 mg/kg in rats (n = 4) are described in Fig. 3. The calculated pharmacokinetic parameters are shown in Table 2. The AUC0–? values were 5.04 ± 0.56, 17.3 ± 1.0, and 51.8 ± 5.3 lg h/mL for 0.5, 2, and 5 mg/kg, respectively. Cmax values were 0.283 ± 0.038, 0.988 ± 0.205, and 2.62 ± 0.76 lg/mL for 0.5, 2, and 5 mg/kg, respectively. Tmax values were 2.33 ± 3.13, 4.00 ± 2.00, and 6.25 ± 4.27 h for 0.5, 2, and 5 mg/kg, respectively. The T values were 10.4 ± 1.5, 8.40 ± 2.46, and 8.96 ± 1.42 h for doses of 0.5, 2, and 5 mg/kg, respectively. Moreover, the %F values were 86.2, 71.8, and 81.3 % for 0.5, 2, and 5 mg/kg, respectively. The
10 5 mg/kg Oral dose 2 mg/kg Oral dose 0.5 mg/kg Oral dose
1
0.1
0.01
0.001 0
8
16
24
32
40
48
56
64
72
81.3
Data from Song et al. (2014)
%F values did not differ statistically among the doses tested (by one-way ANOVA), indicating that the absorption of enzalutamide was also a linear process, like disposition. Following an intravenous dose of enzalutamide in rats, 0.0620 ± 0.0151 and 2.04 ± 0.68 % of the dose was excreted in urine and in feces, respectively, as unchanged drug. These values were 0.0543 ± 0.0204 and 1.83 ± 0.42 %, respectively, after oral administration. These elimination profiles are not significantly different. The renal and fecal clearances of enzalutamide were 0.0497 ± 0.0093 and 1.72 ± 0.57 mL/h/kg, respectively. The tissue concentration–time profiles of enzalutamide after an intravenous injection of 1 mg/kg in rats (n = 3, per time point) are described in Fig. 4. Most tissue maximum concentrations were observed at the first sampling time point of 5 min, except gut and adipose tissue (1 h). The AUC0–72 h vales in 10 tissues (brain, liver, kidneys, testis, heart, lungs, spleen, gut, muscle, and adipose) ranged from 9.03 (brain) to 226 lg h/g (adipose tissue). The ratio of AUC? in the tissues to that in the plasma (Kp) ranged from 0.406 (brain) to 10.2 (adipose tissue) (Table 3). To estimate the in vivo intrinsic clearance of enzalutamide metabolism in the liver, we incubated the material with rat liver microsomes. The proportion of enzalutamide remaining in incubation solution was 98.4 ± 1.9 % after 0.5 h of incubation. The estimated ke value in liver microsomes was 0.000602 ± 0.000515 min-1 in 2 lM and the in vitro intrinsic clearance (CLint, in vitro) 35.58 ± 27.66 mL/h/kg in rat liver. The proportion of the drug that bound to rat plasma protein was 94.7 ± 0.1 % (2 lg/mL enzalutamide).
Discussion
Time (h)
Fig. 3 Plasma concentration–time profiles for enzalutamide in rats after oral administration. Each data point represents the concentration of enzalutamide after oral administration of doses ranging from 0.5 to 5 mg/kg. The use of 2 mg/kg was reported in a previous study (Song et al. 2014)
After intravenous injection (IV, 0.5–5 mg/kg, n = 3) of enzalutamide into rats, the CL was 84.3 mL/h/kg and the Vss was 1140 mL/kg. With oral administration (PO, 0.5–5 mg/kg, n = 4), the F value was 79.7 %. The T was 9.99 h with IV and 9.25 h with PO dosing. There were no
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Tissue concentration of enzalutamide (µg/g)
100
10
Plasma
Heart
Brain
Lung
Liver
Spleen
Kidney
Gut
Testis
Muscle Adipose
1
0.1
0.01
0.001 0
8
16
24
32
40
48
56
64
72
Time (h)
Fig. 4 Tissue concentration–time profiles of enzalutamide in rats after 1 mg/kg intravenous bolus injection
Table 3 Distribution of enzalutamide after intravenous injection (1 mg/kg) and tissue volumes of rat Tissue
AUC (lg/g h)
Kp
Volume (mL)a
Blood
22.3
1.00
54.0
Brain Liver
9.03 124
0.406 5.57
6.80 41.2 9.20
Kidney
67.3
3.03
Testis
14.9
0.671
10.0
Heart
32.5
1.46
3.20
Lung
34.3
1.54
4.00
Spleen
17.2
0.772
2.40
Gut
17.9
0.806
40.0
Muscle
11.7
0.525
488
Adipose
226
10.2
40.0
a
Data from Peters (2012)
statistically significant differences (by one-way ANOVA) among the results. The results suggest that the absorption, distribution, and elimination of enzalutamide occur via linear processes over the dose range studied. All tissue concentrations after an intravenous injection of 1 mg/kg in rats (n = 3, per time point) decreased during the plasma elimination phase and some tissues exhibited a fairly constant tissue-to-plasma ratio. However, the liver-, kidney-, testis-, spleen-, and muscle-to-plasma ratios were increased slightly indicating slower elimination of the drug from these tissues than from plasma. Drug did not accumulate in all tissues at this dose. To confirm the drug distribution, Kp was used, calculated from AUCplasma versus AUCtissue (brain, testis, gut, liver, kidney, spleen, lung, adipose, muscle, and heart) after
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an IV bolus injection of 1 mg/kg. The values ranged from 0.406 (brain) to 10.2 (adipose). Compared with the corresponding plasma concentrations, the liver, kidney, and adipose tissues had higher drug levels. The distributions in brain, testis heart, lung, spleen, gut, and muscle were similar to or lower than plasma. The unusually high amounts of enzalutamide in liver and adipose tissue could be explained of active transport. Using the Kp values and literature sources (Davies and Morris 1993; Peters 2012), we determined the volume of distribution at steady state using the formula, Vss = Vplasma ? R(Vreal tissue 9 Kp, tissue) (Lee et al., 2011). Thus, the calculated Vss was 1028 mL/kg, very close to the Vss estimated from the systemically administered enzalutamide experiment (1135 mL/kg). This indicates that the 10 organs examined are the main distribution organs for enzalutamide. Enzalutamide is approximately 94.7 % bound to plasma protein in the rat. These in vitro protein binding results were highly correlated with the clinical data (97–98 % binding) (Astellas 2014). The recoveries of unchanged enzalutamide were approximately 2 % in feces and less than 0.1 % in urine after intravenous or oral administration (1 mg/kg, n = 3). These results were similar to human clinical data. The recovery of the unchanged form of enzalutamide in humans was only 0.4 % (in feces) (Astellas 2014). The fecal clearance (1.76 mL/h/kg) in the rat was lower than the CL of enzalutamide (84.2 mL/h/kg); thus, fecal excretion was almost negligible. Considering the plasma protein binding and the CLr of enzalutamide, the estimated CLr values for the free fraction (unbound to plasma proteins, 5.29 %) were 0.940 mL/h/kg. This value is lower than the reported glomerular filtration rate in rats (314 mL/h/kg; Davies and Morris 1993). Thus, these results suggest that 99.7 % of enzalutamide is reabsorbed in the renal tubules after glomerular filtration. Although most drugs seem to be eliminated within 78 h after dosing in rats, the amounts excreted unchanged in urine and feces were less than 2 % of the dose administered. Accordingly, the major elimination route of enzalutamide is considered to be not via the urinary and/or fecal routes but via metabolism. In the previous literature, it was reported that enzalutamide is primarily metabolized to N-desmethyl enzalutamide by CYP2C8 and 3A4 in humans. And it was also reported that the half-life of enzalutamide in the presence of human recombinant CYP enzymes including CYP2C8, CYP3A4, and CYP2A5 was [2 h (Astellas 2014). To elucidate the metabolic profile in rats, we incubated enzalutamide with liver microsomes. Enzalutamide was very stable in hepatic microsomes and in vitro half-life calculated using ke value was 19.2 h, which was similar to that from in vivo PK data (9.98 h). However, in vitro hepatic clearance (CLH) calculated using the ‘well stirred’ model
Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats
occupied a little portion in in vivo systemic clearance (1.92 vs. 84.3 mL/h/kg) (Davies and Morris 1993; Pang and Rowland 1977). In this study, we did not directly investigate the underlying reason(s) of discrepancy, but which is likely that enzalutamide was undergo not only Phase I metabolism but also other metabolism. Thus, phase II metabolism of enzalutamide to metabolites such as acyl glucuronides need to be evaluated in the rat because such materials are not observed in humans. We will evaluate the intrinsic clearance of enzalutamide in rat hepatocyes in further studies.
Conclusions Dose-independent pharmacokinetics was observed for enzalutamide in rats after intravenous and oral administration in the dose range 0.5–5 mg/kg. Enzalutamide was mainly distributed to the 10 tissues examined and appeared to be eliminated primarily by metabolism; elimination of the drug in the urine and/or feces was almost negligible. Acknowledgments This work was supported by the research fund of Chungnam National University.
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Kahl, P., L. Gullotti, L.C. Heukamp, S. Wolf, N. Friedrichs, R. Vorreuther, G. Solleder, P.J. Bastian, J. Ellinger, E. Metzger, et al. 2006. Androgen receptor coactivators lysine-specific histone demethylase 1 and four and a half LIM domain protein 2 predict risk of prostate cancer recurrence. Cancer Research 66: 11341–11347. Koo, T.S., S.J. Kim, D.J. Ha, M. Baek, and H. Moon. 2011. Pharmacokinetics, brain distribution, and plasma protein binding of the antiepileptic drug lacosamide in rats. Archives of Pharmacal Research 34: 2059–2064. Lee, K.R., Y.J. Chae, and T.S. Koo. 2011. Pharmacokinetics of lurasidone, a novel atypical anti-psychotic drug, in rats. Xenobiotica; The Fate of Foreign Compounds in Biological Systems 41: 1100–1107. Metzger, E., M. Wissmann, N. Yin, J.M. Muller, R. Schneider, A.H. Peters, T. Gunther, R. Buettner, and R. Schule. 2005. LSD1 demethylates repressive histone marks to promote androgenreceptor-dependent transcription. Nature 437: 436–439. Mullard, A. 2013. 2012 FDA drug approvals. Nature Reviews Drug Discovery 12: 87–90. Nguyen, T.V., M. Yao, and C.J. Pike. 2007. Flutamide and cyproterone acetate exert agonist effects: induction of androgen receptordependent neuroprotection. Endocrinology 148: 2936–2943. Pang, K.S., and M. Rowland. 1977. Hepatic clearance of drugs. I. Theoretical considerations of a ‘‘well-stirred’’ model and a ‘‘parallel tube’’ model. Influence of hepatic blood flow, plasma and blood cell binding, and the hepatocellular enzymatic activity on hepatic drug clearance. Journal of Pharmacokinetics and Biopharmaceutics 5: 625–653. Parkin, D.M., F. Bray, J. Ferlay, and P. Pisani. 2005. Global cancer statistics, 2002. CA: A Cancer Journal for Clinicians 55: 74–108. Peters, S.A. 2012. Physiologically-based pharmacokinetic (PBPK) modeling and simulations: principles, methods, and applications in the pharmaceutical industry. Hoboken: Wiley. Scher, H.I., T.M. Beer, C.S. Higano, A. Anand, M.E. Taplin, E. Efstathiou, D. Rathkopf, J. Shelkey, E.Y. Yu, J. Alumkal, et al. 2010. Antitumour activity of MDV3100 in castration-resistant prostate cancer: a phase 1-2 study. Lancet 375: 1437–1446. Scher, H.I., K. Fizazi, F. Saad, M.E. Taplin, C.N. Sternberg, K. Miller, R. de Wit, P. Mulders, K.N. Chi, N.D. Shore, et al. 2012. Increased survival with enzalutamide in prostate cancer after chemotherapy. New England Journal of Medicine 367: 1187–1197. Semenas, J., N. Dizeyi, and J.L. Persson. 2013. Enzalutamide as a second generation antiandrogen for treatment of advanced prostate cancer. Drug Design Development and Therapy 7: 875–881. Song, J.H., T.H. Kim, J.W. Jung, N. Kim, S.H. Ahn, S.O. Hwang, N.S. Kang, S.E. Yoo, and T.S. Koo. 2014. Quantitative determination of enzalutamide, an anti-prostate cancer drug, in rat plasma using liquid chromatography-tandem mass spectrometry, and its application to a pharmacokinetic study. Biomedical Chromatography 28(8): 1112–1117. Tran, C., S. Ouk, N.J. Clegg, Y. Chen, P.A. Watson, V. Arora, J. Wongvipat, P.M. Smith-Jones, D. Yoo, A. Kwon, et al. 2009. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science 324: 787–790.
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RESEARCH ARTICLE
Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats Tae-Heon Kim1 • Jong-Woo Jeong1 • Ji-Hye Song1 • Kyeong-Ryoon Lee2 Sunjoo Ahn3,4 • Sung-Hoon Ahn3,5 • Sungsub Kim1 • Tae-Sung Koo1
•
Received: 1 September 2014 / Accepted: 30 March 2015 Ó The Pharmaceutical Society of Korea 2015
Abstract We characterized the pharmacokinetics of enzalutamide, a novel anti-prostate cancer drug, in rats after intravenous and oral administration in the dose range 0.5–5 mg/kg. Tissue distribution, liver microsomal stability, and plasma protein binding were also examined. After intravenous injection, systemic clearance, volumes of distribution at steady state (Vss), and half-life (T) remained unaltered as a function of dose, with values in the ranges of 80.4–86.3 mL/h/kg, 1020–1250 mL/kg, and 9.13–10.6 h, respectively. Following oral administration, absolute oral bioavailability was 89.7 % and not dose-dependent. The recoveries of enzalutamide in urine and feces were 0.0620 and 2.04 %, respectively. Enzalutamide was distributed primarily in 10 tissues (brain, liver, kidneys, testis, heart, spleen, lungs, gut, muscle, and adipose) and tissue-to-plasma ratios of enzalutamide ranged from 0.406 (brain) to 10.2 (adipose tissue). Further, enzalutamide was Tae-Heon Kim and Jong-Woo Jeong have been contributed equally to this work. & Sungsub Kim [email protected] & Tae-Sung Koo [email protected] 1
Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon, Korea
2
Life Science Research Institute, Daewoong Pharmaceutical Corporation, Yongin, Korea
3
Center for Drug Discovery Technology, Korea Research Institute of Chemical Technology, Daejeon, Korea
4
Department of Medicinal Chemistry & Pharmacology, Korea University of Science and Technology, Daejeon, Korea
5
College of Pharmacy, Kangwon National University, Chuncheon, Korea
stable in rat liver microsomes, and its plasma protein binding was 94.7 %. In conclusion, enzalutamide showed dose-independent pharmacokinetics at intravenous and oral doses of 0.5–5 mg/kg. Enzalutamide distributed primarily to 10 tissues and appeared to be eliminated primarily by metabolism. Keywords Enzalutamide Pharmacokinetics Tissue distribution Metabolsim Excretion
Introduction Prostate cancer is the most common malignancy and the second leading cause of cancer-related death in men worldwide. The World Health Organization (WHO) has reported the worldwide prostate cancer prevalence as *900,000 diagnosed cases per year, and 239,000 new cases, in the United States in 2013, with a projected death total of 29,700 (Parkin et al. 2005). Prostate cancer develops in the prostate gland of the male reproductive system. The growth of prostate cancer has been demonstrated to be involve androgens and androgen signaling pathways (Huggins and Hodges 2002). Generally, surgical and chemical castration have been used to treat prostate cancer. While surgical castration includes radical orchiectomy (i.e., the removal of the testicles, the source of 95 % of the body’s testosterone), chemical castration may involve administration of various pharmaceuticals such as luteinizing hormone-releasing hormone (LH-RH) agonists, anti-androgens, or other testosterone biosynthesis-inhibiting drugs. Application of androgen deprivation therapy (ADT) may lead to decreased testosterone biosynthesis and result in a reduction in prostate-specific antigen (PSA), tumor degeneration,
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remedy of symptoms, and increased survival of patients (Crawford et al. 1989). Although considerable progress has been made in improving the therapy of prostate cancer, one-third of treated prostate cancer patients will experience disease recurrence and will progress into castration-resistant prostate cancer (CRPC). For these reasons, there is a continuing need for novel drugs that may selectively target the major signaling pathways that contribute to CRPC. Because androgen receptor (AR)-associated pathways are responsible for cancer cell growth and survival, and even enable the cancer cells to grow in the presence of minimal androgen levels (Kahl et al. 2006; Metzger et al. 2005), AR signaling is a useful target for designing and developing such drugs. However, many first-generation antiandrogens, such as flutamide, nilutamide, cyproterone acetate, and bicalutamide, have failed to demonstrate substantial efficacy in CRPC patients. Moreover, these drugs may have AR-agonizing effects (Nguyen et al. 2007). Enzalutamide, previously known as MDV3100 and now available commercially as XtandiÒ, is an anti-prostate cancer drug that is a second-generation AR signaling inhibitor. Enzalutamide blocks the growth of CRPC in cellular model systems and has been shown to increase survival in patients with metastatic CRPC (mCRPC) in a clinical trial (Semenas et al. 2013). Enzalutamide was recently approved (2012) for the treatment of mCRPC postdocetaxel by the US Food and Drug Administration (Mullard 2013; Semenas et al. 2013). Unlike antiandrogens (e.g., bicalutamide), enzalutamide does not have agonist effects in AR signaling but competitively inhibits androgen binding to the AR. Moreover, it inhibits the nuclear translocation of androgens and interactions with DNA. As a result, it induces apoptosis and decreases tumor volume and the proliferation of cancer cells (Scher et al. 2012; Tran et al. 2009). Some clinical pharmacokinetic studies on enzalutamide have been reported (Astellas 2012; El-Amm et al. 2013; Scher et al. 2010), although there limited information describing the pharmacokinetics of enzalutamide in animal models (Astellas 2014; Song et al. 2014). The pharmacokinetics in experimental animals, especially rats, is generally useful as reference data for drug discovery and basic data for analyzing efficacy and toxicity. Also, the evaluation of pharmacokinetics is meaningful for understanding the specific properties (e.g., absorption, distribution, metabolism, and elimination) of drugs in the body. Thus, in the present study, we characterized the pharmacokinetics of enzalutamide after intravenous and oral administration, and evaluated its distribution into major tissues, including the brain, heart, liver, kidney, and testis, in rats.
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Materials and methods Chemicals Enzalutamide was obtained from the Korea Research Institute of Chemical Technology, and bicalutamide, an internal standard, was purchased from Sigma Aldrich. Ammonium acetate salt and other reagents were also from Sigma Aldrich. Acetonitrile and methanol were HPLCgrade and from J.T. Baker. Rat plasma was anticoagulated with sodium heparin, and came from our laboratory. Animals Sprague Dawley (SD) rats (males, 7 weeks old, 204–230 g) were purchased from Orient Bio Inc., (Seongnam, Korea). All animals were kept in an animal room with controlled illumination (12/12 h light/dark cycle), temperature (20–25 °C), and humidity (over 40 %). The animals were fasted for 15 h before dose administration. Animal experiments Intravenous and oral administration of enzalutamide in rats Enzalutamide was dissolved in vehicle (10 % DMSO, 45 % polyethylene glycol 400, and 45 % saline). As a single dose, the administration routes were an intravenous bolus via the tail vein (n = 3) and an oral gavage dose (n = 4). Dosing volume was 1 mL per kg (body weight) and the dosing range was 0.5, 2, and 5 mg/kg. The use of 2 mg/kg was reported in a previous study (Song et al. 2014). A blood sample (200 lL) was collected from the jugular vein using a heparinized syringe to ensure anticoagulation at 0.08 (intravenous only), 0.33, 1, 3, 6, 10, 24, 48, and 72 h after dosing. During blood sampling, rats were placed in a restrainer (Nagai-CFS-1S, NMS, Tokyo, Japan) (Koo et al. 2011). To prepare plasma samples, all blood samples were centrifuged at 13,5009g for 5 min. The samples were stored at -20 °C until analyzed. Determination of urinary and fecal excretion Male SD rats (n = 3) were administered enzalutamide through the tail vein (intravenous) and by oral gavage at 1 mg/kg and were kept in metabolic cages after dosing. Urine and feces samples were collected over the following time intervals after dosing: 0–2, 2–4, 4–6, 6–10, 10–24, 24–48, and 48–72 h. The metabolic cages were rinsed with distilled water, and residues were added to the urine samples at 72 h. To extract the enzalutamide present in the
Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats
feces, samples were shaken vigorously for 12 h with 50 % methanol. The amount of drug excreted in urine or feces (Ae) and the fractions of dose excreted unchanged in urine or feces (fe) were calculated as: Ae ¼Cobs; urine or feces Vurine or feces fe ¼Ae =Dose The urinary and fecal clearances of enzalutamide were calculated as: CLurine or feces ¼ Ae; urine or feces =AUC Estimation of tissue-to-plasma concentration ratio Male SD rats were administered enzalutamide as an intravenous bolus (n = 3, at each time point); dosing conditions were 1 mg/kg (in 10 % DMSO, 45 % polyethylene glycol 400, and 45 % saline) and 1 mL/kg. Animals were sacrificed at 0.08, 0.33, 1, 3, 6, 10, 24, 48, and 72 h after dosing. As biological materials, blood and 10 major tissues (brain, testis, gut, liver, kidney, spleen, lung, adipose, muscle, and heart) were collected. Each tissue was homogenized with the same weight of water (50:50, w/w) using a homogenizer for 5 min at 30 Hz (FB50, Fischer Scientific, USA). The plasma samples and tissue homogenates were stored at -20 °C until analyzed.
In vitro experiments Estimation of hepatic intrinsic clearance of enzalutamide in rat liver microsomes Rat liver microsomes (BD Biosciences, San Jose, CA) were used to estimate the intrinsic clearance of enzalutamide. A typical reaction mixture (500 lL) consisted of rat liver microsomal protein (final concentration = 0.5 mg protein/ mL incubation mixture) and an NADPH regenerating system (final concentrations: 1.3 mM NADP?, 3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, and 3.3 mM magnesium chloride) in 100 mM potassium phosphate buffer (pH 7.4). The mixture was pre-incubated in a water bath at 37 °C for 5 min and an aliquot of enzalutamide solution added to a final concentration of 2 lM. Aliquots (50 lL) of the mixture were sampled at 0, 5, 15, and 30 min after initiation of the reaction. Immediately after collection, a stop solution (100 lL ice-chilled methanol) was added to the sample to terminate the reaction. After vigorous vortexing, and centrifuging at 10,0009g for 5 min, an aliquot (50 lL) of the supernatant was assayed. The concentration of enzalutamide remaining in the sample was plotted against the
reaction time to determine the metabolic rate constant of the reaction. To estimate the in vitro intrinsic clearance in rat livers, that clearance was calculated using the metabolic stability data from rat liver microsomes. The slope (ke) of the temporal profile for the percentage of enzalutamide remaining in a microsomal incubation were obtained by linear regression after logarithmic transformation. Assuming that the substrate concentration used in the experiments (2 lM) was below the KM for the metabolic reaction, the in vitro intrinsic clearance (CLint, in vitro) was calculated as: CLint; in vitro ¼ Aprotein ke =Cprotein where Aprotein is the total amount of liver microsomal protein, and Cprotein the microsomal protein concentration (mg/mL). In this calculation, an Aprotein of 1790 mg protein per kg was used for the rat (Houston and Carlile 1997). Estimation of the fraction of enzalutamide bound to plasma protein We conducted a protein-binding study to determine the fraction of unbound enzalutamide in rat plasma. Binding of test material was assessed via equilibrium dialysis using REDÒ devices (Thermo, Rockford, IL, USA). All assessments were made in triplicate. After 200 lL samples of plasma containing 2 lg/mL enzalutamide were placed into a sample chamber, 350 lL of phosphate buffer (pH 7.4) was added to the buffer chamber. The device containing the samples was incubated at 37 °C for 4 h in a shaking water bath. After incubation, enzalutamide in was assayed in plasma and buffer. Analytical procedure for the determination of enzaluta mide To evaluate enzalutamide levels in the biological samples, a specific high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) (FDA 2001; Song et al. 2014) protocol was used. To induce the precipitation of endogenous proteins, an aliquot (50 lL) of internal standard (IS) solution (bicalutamide 1 lg/mL in acetonitrile) and acetonitrile (200 lL) was added to the biological materials (50 lL; plasma, urine, feces or tissue homogenate). The mixture was mixed vigorously for 10 min, followed by centrifugation (13,5009g, 15 min). An aliquot (5 lL) of the supernatant was injected directly into the HPLC/MS/MS system. The HPLC/MS/MS system consisted of an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) and an API 4000 tandem quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City,
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CA, USA). Compounds were separated on an Agilent Eclipse Plus C18 column (3.5 lm, 2.1 9 50 mm; Agilent Technologies) with an isocratic mobile phase comprising 10 mM ammonium acetate in 40:60 (v/v) water–methanol at a flow rate of 0.3 mL/min. The column and autosampler tray were held at 25 and 4 °C, respectively. The eluent was introduced directly into the tandem quadrupole mass spectrometer through a turbo ion spray source with the following settings: curtain gas, 20 psi; nebulizer gas, 40 psi; turbo gas, 40 psi; ion spray voltage, 5500 V; temperature, 500 °C; declustering potentials, 96, 91 and 101 V; entrance potential, 10 V; collision energies, 47 and 51 V; collision cell exit potentials, 14 and 16 V for EZT and IS, respectively. The analytical run time was 5.0 min. Multiple reaction monitoring (MRM) mode was used for quantification at m/z 465 ? 209 for EZT and m/z 431 ? 217 for the IS. Data were analyzed using Analyst software (version 1.4.1; Applied Biosystems/AB Sciex). The quantification limits for enzalutamide in rat plasma, urine, feces and tissue homogenate were 1, 1, 3 and 3 ng/ mL, respectively. The coefficients of variation of the assay (within and between-day) were generally low (below 10.6 %).
(ANOVA), with Tukey’s test. For the determination of statistically significant correlations, tests of zero correlation were used. In both analyses, p values \0.05 were considered to indicate statistical significance (Fig. 1).
Results Figure 2 describes the mean plasma concentration–time profiles of enzalutamide after intravenous bolus injection of doses in the range 0.5–5 mg/kg in rats (n = 3). The calculated pharmacokinetic parameters are shown in Table 1. The AUC0–? values were 5.85 ± 0.67, 24.2 ± 6.2, and 63.7 ± 12.8 lg h/mL for the 0.5, 2, and 5 mg/kg doses, respectively. The AUC increased linearly as the dose increased. The MRTs were 14.2, 12.0, and 14.7 h for the 0.5, 2, and 5 mg/kg doses, respectively. The CLs were 86.3 ± 10.7, 86.2 ± 19.7, and 80.4 ± 14.5 mL/h/kg for doses of 0.5, 2, and 5 mg/kg, respectively. Vss values were 1250 ± 420, 1020 ± 110, and 1140 ± 70 mL/kg for doses of 0.5, 2, and F F3C
Pharmacokinetic analyses
NH
S
NC
N
N
O Fig. 1 Chemical structure of enzalutamide
Plasma concentration of enzalutamide (µg/mL)
The areas under the plasma concentration–time curve (AUC) and the first moment curve (AUMC) were calculated using the linear trapezoidal method, extrapolated to time = infinity. The terminal half-life (T) was calculated as 0.693/k, where k is the slope of the log-linear portion of the concentration–time profile. The systemic clearance (CL), mean residence time (MRT), and the volume of the distribution at steady state (Vss) were calculated as dose/ AUC, AUMC/AUC, and MRTCL, respectively. The extent of absolute oral bioavailability (F) was estimated by dividing the AUC after oral administration by the AUC after intravenous administration of the respective dose. The peak concentration (Cmax) and the time to reach Cmax (Tmax) were read directly from individual plasma concentration–time profiles. The tissue-to-plasma partition coefficient (Kp) for enzalutamide was calculated by dividing the mean AUCtissue by the mean AUCplasma after administration. To obtain the pharmacokinetic parameters above, all plasma and tissue concentration–time profiles were analyzed using a non-compartmental method with nonlinear least squares regression using the WinNonlin software (ver. 4.1; Pharsight, St. Louis, MO).
O
10 5 mg/kg Intravenous dose 2 mg/kg Intravenous dose 0.5 mg/kg Intravenous dose 1
0.1
0.01
0.001 0
8
16
24
32
40
48
56
64
72
Time (h)
Statistical analyses All data are presented as means ± SDs. Statistical analyses were performed using one-way analysis of variance
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Fig. 2 Plasma concentration–time profiles for enzalutamide in rats after intravenous bolus injection. Each data point represents the concentration of enzalutamide after the intravenous injection of doses ranging from 0.5 to 5 mg/kg
Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats Table 1 Pharmacokinetic parameters of enzalutamide after intravenous injection
Table 2 Pharmacokinetic parameters of enzalutamide after oral administration
Parameter
Parameter
Dose (mg/kg) 0.5
2
a
5
Dose (mg/kg) 0.5
2a
5
AUC (h lg/mL)
5.85 ± 0.67
24.2 ± 6.20
63.7 ± 12.8
AUC (h lg/mL)
5.04 ± 0.56
17.3 ± 1.0
51.8 ± 5.3
T (h)
10.6 ± 2.10
9.13 ± 0.56
10.2 ± 2.2
T (h)
10.4 ± 1.5
8.40 ± 2.46
8.96 ± 1.42
CL (mL/h/kg) MRT (h)
86.3 ± 10.7 14.2 ± 2.9
86.2 ± 19.7 12.0 ± 1.7
80.4 ± 14.5 14.7 ± 3.9
Tmax (h) Cmax (lg/mL)
2.33 ± 3.13 0.28 ± 0.04
4.00 ± 2.00 0.99 ± 0.20
6.25 ± 4.27 2.62 ± 0.76
Vss (mL/kg)
1250 ± 420
1020 ± 110
1140 ± 70
F (%)
86.2
71.8
Data are presented as means ± SDs (n = 3)
Data are presented as means ± SDs (n = 4)
a
a
Data from Song et al. (2014)
Plasma concentration of enzalutamide (µg/mL)
5 mg/kg, respectively, and T values were 10.6 ± 2.1, 9.13 ± 0.56, and 10.2 ± 2.2 h for doses of 0.5, 2, and 5 mg/kg, respectively. There were no significant differences in CL or Vss among the doses (one-way ANOVA), indicating that the elimination and distribution of enzalutamide were linear processes. The plasma concentration–time profiles after oral administration from 0.5 to 5 mg/kg in rats (n = 4) are described in Fig. 3. The calculated pharmacokinetic parameters are shown in Table 2. The AUC0–? values were 5.04 ± 0.56, 17.3 ± 1.0, and 51.8 ± 5.3 lg h/mL for 0.5, 2, and 5 mg/kg, respectively. Cmax values were 0.283 ± 0.038, 0.988 ± 0.205, and 2.62 ± 0.76 lg/mL for 0.5, 2, and 5 mg/kg, respectively. Tmax values were 2.33 ± 3.13, 4.00 ± 2.00, and 6.25 ± 4.27 h for 0.5, 2, and 5 mg/kg, respectively. The T values were 10.4 ± 1.5, 8.40 ± 2.46, and 8.96 ± 1.42 h for doses of 0.5, 2, and 5 mg/kg, respectively. Moreover, the %F values were 86.2, 71.8, and 81.3 % for 0.5, 2, and 5 mg/kg, respectively. The
10 5 mg/kg Oral dose 2 mg/kg Oral dose 0.5 mg/kg Oral dose
1
0.1
0.01
0.001 0
8
16
24
32
40
48
56
64
72
81.3
Data from Song et al. (2014)
%F values did not differ statistically among the doses tested (by one-way ANOVA), indicating that the absorption of enzalutamide was also a linear process, like disposition. Following an intravenous dose of enzalutamide in rats, 0.0620 ± 0.0151 and 2.04 ± 0.68 % of the dose was excreted in urine and in feces, respectively, as unchanged drug. These values were 0.0543 ± 0.0204 and 1.83 ± 0.42 %, respectively, after oral administration. These elimination profiles are not significantly different. The renal and fecal clearances of enzalutamide were 0.0497 ± 0.0093 and 1.72 ± 0.57 mL/h/kg, respectively. The tissue concentration–time profiles of enzalutamide after an intravenous injection of 1 mg/kg in rats (n = 3, per time point) are described in Fig. 4. Most tissue maximum concentrations were observed at the first sampling time point of 5 min, except gut and adipose tissue (1 h). The AUC0–72 h vales in 10 tissues (brain, liver, kidneys, testis, heart, lungs, spleen, gut, muscle, and adipose) ranged from 9.03 (brain) to 226 lg h/g (adipose tissue). The ratio of AUC? in the tissues to that in the plasma (Kp) ranged from 0.406 (brain) to 10.2 (adipose tissue) (Table 3). To estimate the in vivo intrinsic clearance of enzalutamide metabolism in the liver, we incubated the material with rat liver microsomes. The proportion of enzalutamide remaining in incubation solution was 98.4 ± 1.9 % after 0.5 h of incubation. The estimated ke value in liver microsomes was 0.000602 ± 0.000515 min-1 in 2 lM and the in vitro intrinsic clearance (CLint, in vitro) 35.58 ± 27.66 mL/h/kg in rat liver. The proportion of the drug that bound to rat plasma protein was 94.7 ± 0.1 % (2 lg/mL enzalutamide).
Discussion
Time (h)
Fig. 3 Plasma concentration–time profiles for enzalutamide in rats after oral administration. Each data point represents the concentration of enzalutamide after oral administration of doses ranging from 0.5 to 5 mg/kg. The use of 2 mg/kg was reported in a previous study (Song et al. 2014)
After intravenous injection (IV, 0.5–5 mg/kg, n = 3) of enzalutamide into rats, the CL was 84.3 mL/h/kg and the Vss was 1140 mL/kg. With oral administration (PO, 0.5–5 mg/kg, n = 4), the F value was 79.7 %. The T was 9.99 h with IV and 9.25 h with PO dosing. There were no
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Tissue concentration of enzalutamide (µg/g)
100
10
Plasma
Heart
Brain
Lung
Liver
Spleen
Kidney
Gut
Testis
Muscle Adipose
1
0.1
0.01
0.001 0
8
16
24
32
40
48
56
64
72
Time (h)
Fig. 4 Tissue concentration–time profiles of enzalutamide in rats after 1 mg/kg intravenous bolus injection
Table 3 Distribution of enzalutamide after intravenous injection (1 mg/kg) and tissue volumes of rat Tissue
AUC (lg/g h)
Kp
Volume (mL)a
Blood
22.3
1.00
54.0
Brain Liver
9.03 124
0.406 5.57
6.80 41.2 9.20
Kidney
67.3
3.03
Testis
14.9
0.671
10.0
Heart
32.5
1.46
3.20
Lung
34.3
1.54
4.00
Spleen
17.2
0.772
2.40
Gut
17.9
0.806
40.0
Muscle
11.7
0.525
488
Adipose
226
10.2
40.0
a
Data from Peters (2012)
statistically significant differences (by one-way ANOVA) among the results. The results suggest that the absorption, distribution, and elimination of enzalutamide occur via linear processes over the dose range studied. All tissue concentrations after an intravenous injection of 1 mg/kg in rats (n = 3, per time point) decreased during the plasma elimination phase and some tissues exhibited a fairly constant tissue-to-plasma ratio. However, the liver-, kidney-, testis-, spleen-, and muscle-to-plasma ratios were increased slightly indicating slower elimination of the drug from these tissues than from plasma. Drug did not accumulate in all tissues at this dose. To confirm the drug distribution, Kp was used, calculated from AUCplasma versus AUCtissue (brain, testis, gut, liver, kidney, spleen, lung, adipose, muscle, and heart) after
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an IV bolus injection of 1 mg/kg. The values ranged from 0.406 (brain) to 10.2 (adipose). Compared with the corresponding plasma concentrations, the liver, kidney, and adipose tissues had higher drug levels. The distributions in brain, testis heart, lung, spleen, gut, and muscle were similar to or lower than plasma. The unusually high amounts of enzalutamide in liver and adipose tissue could be explained of active transport. Using the Kp values and literature sources (Davies and Morris 1993; Peters 2012), we determined the volume of distribution at steady state using the formula, Vss = Vplasma ? R(Vreal tissue 9 Kp, tissue) (Lee et al., 2011). Thus, the calculated Vss was 1028 mL/kg, very close to the Vss estimated from the systemically administered enzalutamide experiment (1135 mL/kg). This indicates that the 10 organs examined are the main distribution organs for enzalutamide. Enzalutamide is approximately 94.7 % bound to plasma protein in the rat. These in vitro protein binding results were highly correlated with the clinical data (97–98 % binding) (Astellas 2014). The recoveries of unchanged enzalutamide were approximately 2 % in feces and less than 0.1 % in urine after intravenous or oral administration (1 mg/kg, n = 3). These results were similar to human clinical data. The recovery of the unchanged form of enzalutamide in humans was only 0.4 % (in feces) (Astellas 2014). The fecal clearance (1.76 mL/h/kg) in the rat was lower than the CL of enzalutamide (84.2 mL/h/kg); thus, fecal excretion was almost negligible. Considering the plasma protein binding and the CLr of enzalutamide, the estimated CLr values for the free fraction (unbound to plasma proteins, 5.29 %) were 0.940 mL/h/kg. This value is lower than the reported glomerular filtration rate in rats (314 mL/h/kg; Davies and Morris 1993). Thus, these results suggest that 99.7 % of enzalutamide is reabsorbed in the renal tubules after glomerular filtration. Although most drugs seem to be eliminated within 78 h after dosing in rats, the amounts excreted unchanged in urine and feces were less than 2 % of the dose administered. Accordingly, the major elimination route of enzalutamide is considered to be not via the urinary and/or fecal routes but via metabolism. In the previous literature, it was reported that enzalutamide is primarily metabolized to N-desmethyl enzalutamide by CYP2C8 and 3A4 in humans. And it was also reported that the half-life of enzalutamide in the presence of human recombinant CYP enzymes including CYP2C8, CYP3A4, and CYP2A5 was [2 h (Astellas 2014). To elucidate the metabolic profile in rats, we incubated enzalutamide with liver microsomes. Enzalutamide was very stable in hepatic microsomes and in vitro half-life calculated using ke value was 19.2 h, which was similar to that from in vivo PK data (9.98 h). However, in vitro hepatic clearance (CLH) calculated using the ‘well stirred’ model
Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats
occupied a little portion in in vivo systemic clearance (1.92 vs. 84.3 mL/h/kg) (Davies and Morris 1993; Pang and Rowland 1977). In this study, we did not directly investigate the underlying reason(s) of discrepancy, but which is likely that enzalutamide was undergo not only Phase I metabolism but also other metabolism. Thus, phase II metabolism of enzalutamide to metabolites such as acyl glucuronides need to be evaluated in the rat because such materials are not observed in humans. We will evaluate the intrinsic clearance of enzalutamide in rat hepatocyes in further studies.
Conclusions Dose-independent pharmacokinetics was observed for enzalutamide in rats after intravenous and oral administration in the dose range 0.5–5 mg/kg. Enzalutamide was mainly distributed to the 10 tissues examined and appeared to be eliminated primarily by metabolism; elimination of the drug in the urine and/or feces was almost negligible. Acknowledgments This work was supported by the research fund of Chungnam National University.
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